Journal: Bioactive Materials

Article Title: Extracellular vesicles derived from salivary gland stem cells cultured on microwell scaffolds loaded with WNT3A promote the recovery of salivary gland function damaged by radiation via the YWHAZ-PI3K-AKT pathway

doi: 10.1016/j.bioactmat.2025.06.024

Figure Lengend Snippet: Characterization and analysis of EVs derived from sgEpSCs cultured under different conditions. (A) Schematic representation of EV isolation process from the conditioned medium of sgEpSCs, including sequential filtration, PEG precipitation, and ultracentrifugation. (B) NTA of EVs secreted by sgEpSCs cultured in 2D, PLGA-Microwell, and WNT-Microwell conditions, showing particle concentration and size distribution. (C) Particle concentration of EVs smaller than 200 nm, showing 2.8 × 10 9 particles/mL in 2D culture, 4 × 10 9 particles/mL in 3D PLGA-Microwell culture, and 5.8 × 10 9 particles/mL in 3D WNT-Microwell culture. (D) Average number of EVs released per cell in each culture condition: 20 EVs/cell for 2D, 65 EVs/cell for 3D PLGA-Microwell, and 175 EVs/cell for 3D WNT-Microwell. (E) Size distribution analysis of EVs across different culture conditions. No significant difference in size was observed. Data are shown as the mean ± SD. To compare groups, we used one-way ANOVA with Tukey post-hoc test; ∗compared with 2D; $ compared with 3D PLGA . ∗p < 0.05, ∗∗∗p < 0.001, $ p < 0.05, and $$$ p < 0.001. (F) TEM images of sgEpSCs-derived EVs from each culture condition, showing spherical vesicles with sizes ranging from 50 to 150 nm. (G) Western blot analysis of EV-associated markers. Calnexin (negative marker) was detected in 2D-EVs but absent from 3D PLGA -EVs and 3D WNT -EVs. CD9 and CD81 (positive markers) were highly enriched in 3D PLGA -EVs and 3D WNT -EVs.

Article Snippet: Primary antibodies against CD9 (#13403; Cell Signaling Technology, Danvers, MA, USA), CD81 (#56039; Cell Signaling Technology), and calnexin (#2433; Cell Signaling Technology) were used.

Techniques: Derivative Assay, Cell Culture, Isolation, Filtration, Concentration Assay, Western Blot, Marker

Journal: FEBS Open Bio

Article Title: CD9 ‐association with PIP 2 areas is regulated by a CD9 salt bridge

doi: 10.1002/2211-5463.70084

Figure Lengend Snippet: Pearson correlation coefficient (PCC) between PH and CD9 or EWI‐2 analyzed on directly fixed basal plasma membrane sheets. (A) HaCaT cells were double transfected with PH (the pleckstrin homology domain of phospholipase Cδ1 fused to mCherry) and CD9 or EWI‐2 (each fused to monomeric GFP). One day after transfection, membrane sheets were generated, fixed, and imaged. Left column, membrane visualization by the dye TMA‐DPH (displayed using a grayscale lookup table); middle columns, CD9 or EWI‐2 (green lookup table) and PH (magenta lookup table); right, magnified views of overlays from the boxed regions of the middle images. With the exception of the TMA‐DPH images (left), images of one channel are shown at the same settings of brightness and contrast. (B) The average PCC between PH and CD9 or EWI‐2 analyzed in squared regions of interest (ROIs) placed onto central membrane areas devoid of holes or large fluorescent accumulations. Values are given as means ± SD ( n = 6 biological replicates; each replicate and condition includes 3–13 analyzed membrane sheets; four out of the six replicates are analyzed separately in Figs and ). Statistical test: unpaired two‐tailed Student's t ‐test (ns: non‐significant).

Article Snippet: Then, in a first round of immunolabeling, the membrane was incubated with CD9 primary antibody (1 : 1000 in Intercept blocking buffer) overnight at 4 °C, followed by the secondary antibody IRDye 800CW goat anti‐mouse (1 : 5000 in Intercept blocking buffer) for 1 h at room temperature, followed by imaging using a ChemiDoc MP Imaging System (Bio‐Rad Laboratories, Inc., Hercules, California, USA).

Techniques: Clinical Proteomics, Membrane, Transfection, Generated, Two Tailed Test

Journal: FEBS Open Bio

Article Title: CD9 ‐association with PIP 2 areas is regulated by a CD9 salt bridge

doi: 10.1002/2211-5463.70084

Figure Lengend Snippet: Pearson correlation coefficient (PCC) between EWI‐2 and CD9, CD9‐K11A, CD9‐E84A, or CD9‐K11E/E84K analyzed on directly fixed basal membrane sheets. (A) HaCaT cells were double transfected with EWI‐2 (fused to monomeric RFP) and CD9, CD9‐K11A, CD9‐E84A, or CD9‐K11E/E84K (each fused to monomeric GFP). One day after transfection, membrane sheets were generated, fixed, and imaged. Left column, visualization of membranes by the dye TMA‐DPH (displayed with a grayscale lookup table); middle columns, CD9, CD9‐K11A, CD9‐E84A, or CD9‐K11E/E84K (green lookup table) and EWI‐2 (magenta lookup table); right column, magnified views show overlays from the boxed regions of the middle images. With the exception of the TMA‐DPH images (left column), images of one channel are shown at the same settings of brightness and contrast. (B) PCC between EWI‐2 and CD9, CD9‐K11A, CD9‐E84A, or CD9‐K11E/E84K analyzed with squared regions of interest (ROIs). Values are given as means ± SD ( n = 3 biological replicates; each replicate and condition includes 14–29 analyzed membrane sheets). Statistical test: one‐way ANOVA test followed by Dunnett's multiple comparisons test comparing in (B) CD9 to CD9‐K11A, CD9‐E84A, and CD9‐K11E/E84K (ns: non‐significant; * P < 0.05; ** P < 0.01).

Article Snippet: Then, in a first round of immunolabeling, the membrane was incubated with CD9 primary antibody (1 : 1000 in Intercept blocking buffer) overnight at 4 °C, followed by the secondary antibody IRDye 800CW goat anti‐mouse (1 : 5000 in Intercept blocking buffer) for 1 h at room temperature, followed by imaging using a ChemiDoc MP Imaging System (Bio‐Rad Laboratories, Inc., Hercules, California, USA).

Techniques: Membrane, Transfection, Generated

Journal: FEBS Open Bio

Article Title: CD9 ‐association with PIP 2 areas is regulated by a CD9 salt bridge

doi: 10.1002/2211-5463.70084

Figure Lengend Snippet: Pearson correlation coefficient (PCC) between CD9‐RFP and CD9‐GFP or CD9‐E84A‐GFP. (A) HaCaT cells were double transfected with RFP‐labelled CD9 and GFP‐labelled CD9 or CD9‐E84A. One day after transfection, membrane sheets were generated, fixed, and imaged. Left, visualization of membranes by TMA‐DPH (grayscale lookup table); two middle columns, GFP‐labelled CD9 or CD9‐E84A (left, green lookup table) and RFP‐labelled CD9 (right, magenta lookup table); right, magnified views show overlays from the boxed regions of the middle images. With the exception of the TMA‐DPH images (left), images of one channel are shown at the same settings of brightness and contrast. (B) PCC between RFP‐labelled CD9 and GFP‐labelled CD9 or CD9‐E84A analyzed with squared regions of interest (ROIs). Values are given as means ± SD ( n = 3 biological replicates; each replicate and condition includes 23–31 analyzed membrane sheets). Statistical test: unpaired two‐tailed Student's t ‐test (* P < 0.05).

Article Snippet: Then, in a first round of immunolabeling, the membrane was incubated with CD9 primary antibody (1 : 1000 in Intercept blocking buffer) overnight at 4 °C, followed by the secondary antibody IRDye 800CW goat anti‐mouse (1 : 5000 in Intercept blocking buffer) for 1 h at room temperature, followed by imaging using a ChemiDoc MP Imaging System (Bio‐Rad Laboratories, Inc., Hercules, California, USA).

Techniques: Transfection, Membrane, Generated, Two Tailed Test

Journal: FEBS Open Bio

Article Title: CD9 ‐association with PIP 2 areas is regulated by a CD9 salt bridge

doi: 10.1002/2211-5463.70084

Figure Lengend Snippet: Antibody‐induced patching of CD9, CD9‐K11A and CD9‐E84A. (A) HaCaT cells were double transfected with PH and GFP‐labelled CD9 (top), CD9‐K11A (middle), or CD9‐E84A (bottom). One day after transfection, membrane sheets were generated and either directly fixed (left panels) or after a 5‐min incubation at RT with control buffer (Control, middle panels) or an anti‐GFP antibody (αGFP, right panels). A green lookup table is used for CD9, CD9‐K11A, and CD9‐E84A, and a magenta lookup table for PH. Images of one channel are shown at the same settings of brightness and contrast. In parallel, we performed control experiments: (i) employing the GFP antibody on membrane sheets of cells only expressing PH or (ii) employing different polyclonal antibodies (Fig. ). (B) The average membrane‐associated PH (mCherry‐intensity) analyzed with squared regions of interest (ROIs). The inset shows the αGFP condition after normalization of CD9‐K11A and CD9‐E84A to CD9. (C) PCCs analyzed between PH and CD9, CD9‐K11A, and CD9‐E84A with squared ROIs. Values are given as means ± SD ( n = 3 biological replicates; each replicate and condition includes 13–28 analyzed membrane sheets). Statistical test: two‐way ANOVA test followed by Šidák's multiple comparisons test comparing in (B) and (C) control to directly fixed or αGFP (ns: non‐significant; **** P < 0.0001). (B) Inset, the confidence interval was calculated for the comparison of CD9‐K11A to CD9 (** P < 0.01) or CD9‐E84A to CD9 (** P < 0.01).

Article Snippet: Then, in a first round of immunolabeling, the membrane was incubated with CD9 primary antibody (1 : 1000 in Intercept blocking buffer) overnight at 4 °C, followed by the secondary antibody IRDye 800CW goat anti‐mouse (1 : 5000 in Intercept blocking buffer) for 1 h at room temperature, followed by imaging using a ChemiDoc MP Imaging System (Bio‐Rad Laboratories, Inc., Hercules, California, USA).

Techniques: Transfection, Membrane, Generated, Incubation, Control, Expressing, Comparison

Journal: FEBS Open Bio

Article Title: CD9 ‐association with PIP 2 areas is regulated by a CD9 salt bridge

doi: 10.1002/2211-5463.70084

Figure Lengend Snippet: Antibody‐induced patching of CD9, CD9‐E84A, and EWI‐2. (A) HaCaT cells were double transfected with PH and GFP‐labelled CD9 (top), CD9‐E84A (middle), or EWI‐2 (bottom). One day after transfection, membrane sheets were generated and either directly fixed (left panels) or after a 5‐min incubation at RT without (Control, middle panels) or with an anti‐GFP antibody (αGFP, right panels). A green lookup table is used for CD9, CD9‐E84A, and EWI‐2, and a magenta lookup table for PH. Images of one channel are shown at the same settings of brightness and contrast. (B) The membrane‐associated PH (mCherry‐intensity) was analyzed with squared regions of interest (ROIs). The inset shows the condition αGFP after normalization of CD9‐E84A and EWI‐2 to CD9. (C) PCCs between PH and CD9, CD9‐E84A, and EWI‐2 were analyzed with squared ROIs. Values are given as means ± SD ( n = 4 biological replicates; each replicate and condition includes 10–31 analyzed membrane sheets). Statistical test: two‐way ANOVA test followed by Šidák's multiple comparisons test comparing in (B) and (C) control to directly fixed or αGFP (ns: non‐significant; *** P < 0.001; **** P < 0.0001). (B) Inset, the confidence interval was calculated for the comparison of CD9‐E84A to CD9 (* P < 0.05) or EWI‐2 to CD9 (** P < 0.01).

Article Snippet: Then, in a first round of immunolabeling, the membrane was incubated with CD9 primary antibody (1 : 1000 in Intercept blocking buffer) overnight at 4 °C, followed by the secondary antibody IRDye 800CW goat anti‐mouse (1 : 5000 in Intercept blocking buffer) for 1 h at room temperature, followed by imaging using a ChemiDoc MP Imaging System (Bio‐Rad Laboratories, Inc., Hercules, California, USA).

Techniques: Transfection, Membrane, Generated, Incubation, Control, Comparison

Journal: FEBS Open Bio

Article Title: CD9 ‐association with PIP 2 areas is regulated by a CD9 salt bridge

doi: 10.1002/2211-5463.70084

Figure Lengend Snippet: Antibody‐induced patching of CD9 and EWI‐2 after elevation of PIP 2 . HaCaT cells were double transfected with PH and GFP‐labelled CD9 or EWI‐2, and one day after transfection, membrane sheets were generated. When indicated, prior to membrane sheet generation, cells were incubated for 15 min with 300 m m sucrose. Then, cells were either directly fixed or after a 5‐min incubation at RT without (Control) or with an anti‐GFP antibody (αGFP). (A) The average membrane‐associated PH (mCherry‐intensity) analyzed with squared regions of interest (ROIs). (B) PCCs were analyzed between PH and CD9 or EWI‐2 with squared ROIs. Values are given as means ± SD ( n = 3 biological replicates; each replicate and condition includes 12–43 analyzed membrane sheets). Statistical test: two‐way ANOVA test followed by Šidák's multiple comparisons test comparing in (A) and (B) control to directly fixed or αGFP (ns: non‐significant; * P < 0.05; **** P < 0.0001).

Article Snippet: Then, in a first round of immunolabeling, the membrane was incubated with CD9 primary antibody (1 : 1000 in Intercept blocking buffer) overnight at 4 °C, followed by the secondary antibody IRDye 800CW goat anti‐mouse (1 : 5000 in Intercept blocking buffer) for 1 h at room temperature, followed by imaging using a ChemiDoc MP Imaging System (Bio‐Rad Laboratories, Inc., Hercules, California, USA).

Techniques: Transfection, Membrane, Generated, Incubation, Control

Journal: FEBS Open Bio

Article Title: CD9 ‐association with PIP 2 areas is regulated by a CD9 salt bridge

doi: 10.1002/2211-5463.70084

Figure Lengend Snippet: Relationship between retained PH and the expression level of GFP‐tagged CD9, CD9‐K11A, and CD9‐E84A. For individual membrane sheets analyzed in Fig. , condition αGFP, the intensity of mCherry (retained PH) is plotted versus the GFP intensity (expression level). For CD9, CD9‐K11A, and CD9‐E84A, the average GFP intensity was 4313 a.u. (55 membranes collected from 3 biological replicates), 2988 a.u. (58 membranes from 3 biological replicates) and 2375 a.u. (66 membranes from 3 biological replicates), respectively. Linear regression lines are fitted through the origin. R values and slopes (θ) are stated in the upper right. Open circles, CD9; open diamonds, CD9‐K11A; open squares, CD9‐E84A.

Article Snippet: Then, in a first round of immunolabeling, the membrane was incubated with CD9 primary antibody (1 : 1000 in Intercept blocking buffer) overnight at 4 °C, followed by the secondary antibody IRDye 800CW goat anti‐mouse (1 : 5000 in Intercept blocking buffer) for 1 h at room temperature, followed by imaging using a ChemiDoc MP Imaging System (Bio‐Rad Laboratories, Inc., Hercules, California, USA).

Techniques: Expressing, Membrane

Journal: FEBS Open Bio

Article Title: CD9 ‐association with PIP 2 areas is regulated by a CD9 salt bridge

doi: 10.1002/2211-5463.70084

Figure Lengend Snippet: Relationship between retained PH and the expression level of GFP‐tagged CD9, CD9‐E84A, and EWI‐2. For individual membrane sheets analyzed in Fig. (condition αGFP), the intensity of mCherry (retained PH) is plotted versus the GFP intensity (expression level). For CD9, CD9‐E84A, and EWI‐2, the average GFP intensity was 4865 a.u. (96 membranes collected from 4 biological replicates), 4278 a.u. (93 membranes from 4 biological replicates), and 1269 a.u. (66 membranes from 4 biological replicates), respectively. Regression lines are fitted through the origin. R 2 values and slopes (θ) are stated in the lower right. Open circles, CD9; open squares, CD9‐E84A; open triangles, EWI‐2.

Article Snippet: Then, in a first round of immunolabeling, the membrane was incubated with CD9 primary antibody (1 : 1000 in Intercept blocking buffer) overnight at 4 °C, followed by the secondary antibody IRDye 800CW goat anti‐mouse (1 : 5000 in Intercept blocking buffer) for 1 h at room temperature, followed by imaging using a ChemiDoc MP Imaging System (Bio‐Rad Laboratories, Inc., Hercules, California, USA).

Techniques: Expressing, Membrane

Journal: FEBS Open Bio

Article Title: CD9 ‐association with PIP 2 areas is regulated by a CD9 salt bridge

doi: 10.1002/2211-5463.70084

Figure Lengend Snippet: Opening of CD9 diminishes co‐immunoprecipitated endogenous CD9. (A) HaCaT cells were transfected with GFP‐tagged CD9 or CD9‐E84A. One day after transfection, cells were lysed in 1% Brij97 (input shows the lysate) and the GFP‐tag was pulled down via GFP‐trap beads (IP, immunoprecipitate). Untransfected cells were used as control. Proteins were separated by electrophoresis (SDS‐PAGE), transferred onto a nitrocellulose membrane, followed by the detection of CD9 (lower membrane) and GFP (upper membranes). CD9‐ and GFP western blots are shown at different settings of brightness and contrast. Left and right images (separated by a dashed line) are from the same membrane and shown at the same brightness and contrast settings. Please note that on the lower membrane both overexpressed and endogenous CD9 are detected by the CD9 antibody, whereas on the upper membrane, only the overexpressed GFP‐tagged CD9/CD9‐E84A is detected. (B) The lower membrane in (A) is shown at a different brightness and contrast setting for better illustration of the difference in the bands of co‐precipitated endogenous CD9. (C) The immunoprecipitation efficiency is determined from the IP membranes. Co‐immunoprecipitated endogenous CD9 (CD9‐antibody detection, lower CD9endo band) is related to the band of the respective immunoprecipitated GFP (GFP‐antibody detection, CD9‐GFP/CD9‐E84A‐GFP). For each replicate, the ratio of CD9endo/CD9‐GFP and CD9endo/CD9‐E84A‐GFP was calculated and normalized to CD9endo/CD9‐GFP (set to 100%). Values are given as means ± SD ( n = 4 biological replicates). Statistical test: (C) the confidence interval was calculated for the comparison of CD9‐E84A to CD9 (* P < 0.05).

Article Snippet: Then, in a first round of immunolabeling, the membrane was incubated with CD9 primary antibody (1 : 1000 in Intercept blocking buffer) overnight at 4 °C, followed by the secondary antibody IRDye 800CW goat anti‐mouse (1 : 5000 in Intercept blocking buffer) for 1 h at room temperature, followed by imaging using a ChemiDoc MP Imaging System (Bio‐Rad Laboratories, Inc., Hercules, California, USA).

Techniques: Immunoprecipitation, Transfection, Control, Electrophoresis, SDS Page, Membrane, Western Blot, Comparison

Journal: FEBS Open Bio

Article Title: CD9 ‐association with PIP 2 areas is regulated by a CD9 salt bridge

doi: 10.1002/2211-5463.70084

Figure Lengend Snippet: CD9 opening does not change the distance of CD9 to the next neighbored CD9. (A) After the imaging of membrane sheets by epi‐fluorescence microscopy (Fig. ), membranes were incubated with GFP and RFP nanobodies coupled to dyes enabling super‐resolution imaging by STED microscopy. Magenta and green images show STED micrographs recording the distribution of CD9‐RFP and CD9‐GFP/CD9‐E84A‐GFP, respectively. Middle images are overlays of magnified views of the boxed regions in the left images. Right, magnified views of the boxed regions of the middle images. Maxima are detected in the CD9‐RFP channel (white pixels in the right magenta images) and classified as nano‐clusters (encircled white pixels; for details see text). For each nano‐cluster in the magenta image, the distance to the next nearest maximum in the green image (white pixels mark all maxima) was determined. Numbers in the right green images state the determined shortest distances in nanometers. Middle, white pixels mark maxima of the green image, and arrows mark the positions of the encircled nano‐clusters in the magenta images. (B) Average shortest distance of CD9 to CD9 (left) or CD9 to CD9‐E84A (right), compared with the shortest distance in flipped images (used as control for obtaining the purely random distance). Values are given as means ± SD ( n = 3 biological replicates; each replicate and condition includes 10–17 analyzed membrane sheets). Statistical test: unpaired two‐tailed Student's t ‐test between CD9 and CD9 Flipped (left, * P < 0.05) or CD9‐E84A and CD9‐E84A Flipped (right, ** P < 0.01).

Article Snippet: Then, in a first round of immunolabeling, the membrane was incubated with CD9 primary antibody (1 : 1000 in Intercept blocking buffer) overnight at 4 °C, followed by the secondary antibody IRDye 800CW goat anti‐mouse (1 : 5000 in Intercept blocking buffer) for 1 h at room temperature, followed by imaging using a ChemiDoc MP Imaging System (Bio‐Rad Laboratories, Inc., Hercules, California, USA).

Techniques: Imaging, Membrane, Fluorescence, Microscopy, Incubation, Control, Two Tailed Test

Journal: FEBS Open Bio

Article Title: CD9 ‐association with PIP 2 areas is regulated by a CD9 salt bridge

doi: 10.1002/2211-5463.70084

Figure Lengend Snippet: CD9 distribution in the membrane depends on the state of the salt bridge. In CD9, a salt bridge forms between a negatively charged glutamate of the SIL and a positively charged lysine of the N‐terminus (see magnified view of the closed salt bridge; the red circle with a plus sign and the green circle with a minus sign represent a lysine and a glutamate residue, respectively). In the closed conformation, CD9 prefers PIP 2 ‐enriched areas. In contrast, CD9 lacking the salt bridge prefers areas in which PIP 2 is depleted, like the CD9‐interaction partner EWI‐2. A transfer of open‐CD9 to PIP 2 ‐depleted areas would promote the complex formation between CD9 and EWI‐2.

Article Snippet: Then, in a first round of immunolabeling, the membrane was incubated with CD9 primary antibody (1 : 1000 in Intercept blocking buffer) overnight at 4 °C, followed by the secondary antibody IRDye 800CW goat anti‐mouse (1 : 5000 in Intercept blocking buffer) for 1 h at room temperature, followed by imaging using a ChemiDoc MP Imaging System (Bio‐Rad Laboratories, Inc., Hercules, California, USA).

Techniques: Membrane, Residue

Journal: Cancers

Article Title: Molecular Profiling of A549 Cell-Derived Exosomes: Proteomic, miRNA, and Interactome Analysis for Identifying Potential Key Regulators in Lung Cancer

doi: 10.3390/cancers16244123

Figure Lengend Snippet: Physicochemical, morphological, and biochemical characterization of A549 cell-derived exosomes concentrated via ultrafiltration and isolated using the membrane-based affinity binding method (exoEasy Maxi Kit, QIAGEN). ( a ) DLS analysis of exosomes considering the parameter of volume. The curve represents the means from three successive DLS measurements (n = 3, biological replicates). ( b ) Representative zeta-potential distribution of the exosomes isolated via the proposed methodology. The curves obtained by three successive measurements (n = 3, technical replicates) are depicted. ( c – e ) Cryo-TEM analysis of isolated exosomes. Round-shaped exosomes are depicted (black arrows) with distinct exosomal lipid bilayer (white arrows). ( f ) Detection of CD9 and CD63 exosomal markers as well as cytoplasmic marker β-actin via Western blot analysis. The uncropped bolts are shown in . Equal amounts of protein (30 μg) from exosomes and whole cell lysate as calculated by BCA protein assay kit were loaded in wells of 15% SDS-PAGE gel for separation, followed by Western blotting. ( g ) Flow cytometry-based histogram representing the negative control-beads incubated with the anti-CD9 PE antibody without the addition of exosomes (blue curved), as well as the A549 cell-derived exosomes captured by the beads with subsequent incubation with the anti-CD9 antibody (red curve). The dot plots represent the gating strategies for the exosome containing samples and negative control-beads. Data were analyzed using FlowJo (FlowJo™ Software Ashland: Becton, Dickinson and Company, v10.10.0 (2023) .

Article Snippet: The immunoblots were then incubated with the appropriate primary monoclonal antibody against CD9 or CD63 exosomal marker (1:5000 dilution of CD9/60232-1-Ig and CD63/67605-1-lg Monoclonal antibodies, Proteintech Group, Rosemont, IL, USA) overnight at RT.

Techniques: Derivative Assay, Isolation, Membrane, Binding Assay, Zeta Potential Analyzer, Marker, Western Blot, Bicinchoninic Acid Protein Assay, SDS Page, Flow Cytometry, Negative Control, Incubation, Software